CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation

ABSTRACT RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA engineering begins with local helix distortion by transient CRISPR-Cas protein binding.

The precise mechanism by which Cas12a locates target sequences amidst the vast excess of non-target sites in a typical genome remains unclear.Structural studies of both Cas9 and Cas12a revealed conformational changes that accompany R-loop formation but did not identify the initial steps of DNA engagement (6,7).Structural and biochemical studies of Cas9guide RNA in complex with PAM-containing but otherwise non-target DNA provided evidence for DNA bending causing transient melting of the DNA sequence immediately adjacent to PAMs, enabling initial RNA-guided sequence interrogation (8).Because Cas9 and Cas12 evolved independently from different ancestral proteins (9,10), it has been unclear whether Cas12a functions by a similar mechanism (Fig. 1A).
We used cryo-electron microscopy (cryo-EM) to determine structures of a complex between Acidaminococcus sp.Cas12a-guide RNA and a PAM-containing non target DNA molecule.Using site-specific cross-linking to trap the otherwise transient encounter between the Cas12a ribonucleoprotein (RNP) and the DNA, we observed three classes of DNA conformations with different degrees of bending relative to a standard B-form helix.In the most distorted class, a PAM-proximal target strand nucleotide is unstacked and dynamic, an observation supported by fluorescence-based measurements of DNA conformation in solution.
These data suggest that DNA bending induces base-flipping to enable Cas12a RNA-guided target recognition, a mechanism analogous to that of Cas9 despite independent evolutionary origins (8).Our findings help explain how Cas12a identifies target sites within genomes, a process that influences both the rate and outcomes of Cas12a-mediated genome editing.

DNA oligonucleotide preparation
The cystamine-functionalized DNA oligonucleotide (5'-GTAGTGXTTTGTCGTCTCATCTGTATGCGTC, where X denotes the N4-cystamine-2'deoxycytidine) was synthesized by TriLink Biotechnologies.All other oligonucleotides were obtained from Integrated DNA Technologies.DNA oligonucleotides were purified in-house on urea-PAGE gels.DNA duplexes were annealed in water by heating to 95°C for 2 min and then cooling to 25°C over a 40 min period.

Complex preparation
For crosslinking optimization, 6 µM functionalized DNA duplex, 5 µM RNA, and 4 µM AsCas12a were combined in cross-linking buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 5% glycerol, 100 µM DTT).The reaction was incubated at 25°C for 12 hr and then quenched by addition of S-methyl methanethiolsulfonate to a final concentration of 20 mM.Reactions were then denatured by adding non-reducing SDS loading solution and heating for 5 min at 90°C.
For cryo-EM sample preparation, reactions were prepared as above but without quenching.

EM grid preparation and data collection
Sample aliquots were thawed and diluted into complex buffer to a concentration of 3 µM.Grids
After motion correction (Patch Motion) and CTF estimation (Patch CTF), 1,364 micrographs were curated for further processing.Initial particle picking was done using blob picker on a small subset of micrographs to generate templates that were used (Template Picker) to pick 1,929,565 particles from all micrographs.After two rounds of 2D classification, 459,736 particles remained.Particles were re-extracted with re-centering and used for ab initio reconstruction into 5 classes.Only one class corresponded to the expected complex (137,851 particles).Particles from this class were again re-extracted with re-centering, and subjected to non-uniform refinement, which provided a map with global resolution of 3 Å.In the sharpened map the DNA density was absent.Particles from the non-uniform refinement were transferred to RELION v. 5.0-beta (12) for DNA-focused refinement.After 3D refinement in RELION, signal from the protein was subtracted, the particles were re-centered on DNA, and five DNA volume classes were reconstructed.Three classes, with 20,031, 27,147 and 27,031 particles respectively, were chosen for final reconstruction.Particle subtraction was reversed to yield reconstructions of the full particles.Reconstructions were refined with masking, solvent-flattened FSC and Blush regularization.Refined maps were sharpened (B-factor -30), reaching resolutions 3.6 Å for structure 1, 3.4 Å for structure 2 and 3.5 Å for structure 3.These maps were used for model building and refinement.

Model building
The AsCas12a crystal structure (PDB 5B43) was used as an initial model.In Coot (v.0.9.8.93 EL) (13) each protein domain was fit separately into the density with manual adjustments.Both sharpened and unsharpened maps were used in this process.The protein model was iteratively refined in Phenix (v.1.19.2-4158)(14-16) using phenix.real_space_refine(minimization_global, local_grid_search, adp) with secondary structural restraints and manual geometry adjustments in Coot.Regions where neither the sharpened nor unsharpened map provided sufficient density remained unmodeled.Overall, the REC domain had the weakest density in the structures.The RNA sequence was adjusted to reflect our construct sequence.DNA was created as a linear Bform helix in Coot.PHENIX (16) simulated_annealing with base pair and base stacking restraints was used to fit the DNA into the density.In one of the structures, the base near the PAM sequence was modeled as unstacked and rotated away from the helix axis.UCSF Chimera X ISOLDE (17) was used to improve the model of the DNA, as well as the PI and REC domains of Cas12a.Planarity was enforced on nucleotides within the DNA duplex in the protospacer region (5 base pairs; chain C/D bases 14-18) for all structures.The thioalkane linker was added as an ethanethiol ligand from PHENIX's eLBOW module (18).Sulfur-sulfur and carbon-nitrogen bonds were restrained as described (8).

2-Aminopurine assay to detect unstacked nucleotides
Target strand oligonucleotides including a 2-aminopurine base in position 1 (5'-GACGCATACAGATGAGACGACAAAGCACTAC-3′ -bold font shows the modification insertion, underlined nucleotides are the PAM), position 2 (5'-GACGCATACAGATGAGACGACAAAGCACTAC-3′), position 4 (5'-GACGCATACAGATGAGACGACAAAGCACTAC-3′) and position 13 (5'-GACGCATACAGATGAGACGACAAAGCACTAC-3′) downstream of the PAM sequence in the protospacer region, were obtained from IDT.Reactions (in triplicate, 25°C) were assembled and measured every 75 s over 2 hours using a Biotek Cytation 5 imaging reader with excitation wavelength 320 nm and emission wavelength 370 nm.Reading at 75 sec point was used for analysis.Reading of triplicates were averaged together.All measurements were normalized to the signal from double stranded DNA alone and measurement error was propagated using the following formula: where  is the average of Cas12a-RNA-DNA or Cas12a-DNA signal;  is the average of DNA only signal;   is a standard deviation for Cas12a-RNA-DNA or Cas12a-DNA signal;   is a standard deviation for DNA only signal.

DNA bend and twist calculations
We quantified the local DNA bending and unwinding simultaneously, using an established interhelical Euler angle approach (19,20).This method measures the bending magnitude (β_h, 0° ≤ β_h ≤ 180°), bending direction (γ_h, -180° ≤ γ_h ≤ 180°), and helical twist (ζ_h, -180° ≤ ζ_h ≤ 180°) between two helices (H1 and H2) across a junction (J) containing one or multiple base pairs.We defined the PAM sequences as H1, the spacer sequences two base pairs away from PAM as H2 and the two base pairs immediately adjacent to PAM in the spacer as J. Two idealized B-form DNA helices, each comprising 3 base pairs, were constructed using the 3DNA software (21) and superimposed onto H1 and H2, respectively.This alignment enabled us to determine the relative orientation of H1 to H2, quantified by the parameters β_h, γ_h, and ζ_h.
The underwinding angle of the helix was calculated by N x 36° -ζ_h, where N is the number of base pairs in the J.Using this method, bending angle for Cas9 interrogation complex with bent DNA and closed protein conformation had a bend angle ~69° and unwinding of ~66°, while the linear DNA conformation in an open protein conformation had a bend angle of ~38° and unwinding of ~16°.

Figure preparation
The comparison of PI domains was conducted after structure alignment with SSM superpose in Coot using protein chain.For figure visualization models were aligned with Chimera X (1.7.1) (22) Matchmaker using protein chain in the reference for alignment.DNA-protein contacts were listed with Chimera X "contacts" command.Figures were prepared with Chimera X (1.7.1).
Chromatogram of complex purification and 2-AP assay results were plotted using GraphPad Prism 8.

Cryo-EM structures of a crosslink-stabilized Cas12a:guide RNA:non target DNA complex
To investigate target search by a Cas12a-guide RNA complex, we designed DNA substrates bearing a 5'-TTTG-3' PAM sequence but lacking any base pairing complementarity with the guide RNA (Fig. 1B).We captured transient Cas12a RNP association with this substrate by introducing a disulfide crosslink between mutated amino acid N551C in Cas12a and an N4cystamine-cyt(7) DNA modification.Control experiments confirmed that N551C mutation in Cas12a did not disrupt its ability to cleave target DNA (Fig. S1A).Formation of the disulfide crosslink between Cas12a and the modified DNA was confirmed by denaturing, non-reducing SDS-PAGE analysis (Fig. S1B).
Cryo-EM analysis of the cross-linked sample revealed three distinct molecular structures (Fig. S1C, S2, Table 1).Conformations of the protein in each model are similar to each other and resemble Cas12a-guide RNA binary complexes (PDB ID 5NG6, 5ID6).In all structures, the PAM sits in the Cas12a PAM-binding pocket (Fig. 1C).EM maps show clear density corresponding to the crosslinking disulfide bridge between the protein and the DNA (Fig. 1D).
Consistent with the flexibility of the recognition (REC) lobe in both Cas12a and Cas9 noted in prior studies (6-8, 23, 24), we observed poor EM density for the REC domain in the structures determined here.

Movement of PAM interaction domain induces DNA bending
We analyzed Cas12a-DNA interactions in our cryoEM structures to assess their similarity to prior Cas12a complexes containing target-engaged Cas12a RNPs (2,7,25).In all structures, the DNA sits in a positively charged groove of PI and WED domains.In structures 2 and 3, Pro599, Met604 and Lys607 of the PAM-interacting (PI) domain and Lys548 of the Wedge (WED-II) domain form contacts with the PAM directly adjacent to the crosslink position (Fig. 2A) (2,3).In structure 1, key interactions between Lys548 and the PAM are absent.N7 of adenosine (position -2) on the target strand is 4.46 Å away from Lys548 side chain, placing it out of range for hydrogen bonding.The majority of protein-DNA contacts surround PAM interaction residues and form non-specific interactions with the DNA backbone.This is consistent with previously published molecular structures of the ternary complex of AsCas12a determined by X-ray crystallography (PDB ID 5B43) and cryoEM (PDB ID 8SFH).
The C-alpha alignment of all three molecular models shows the PI domain of structure 2

Cas12a bends DNA to induce helical distortion and base flipping for DNA interrogation
We quantified the relative distortion of DNA in crosslinked structures (Fig. 3A).DNA in the candidate protospacer region is progressively bend and underwound from structures 1 to 3.
In structure 3, which displays a DNA bend of 63° and is underwound by 41°, we observe base unstacking and flipping (Fig. 3B-E).The nucleobase in position 2 downstream of the PAM within the candidate protospacer region is rotated out of its normal DNA base-pairing position (Fig. 3A, E; Fig. S3).The corresponding nucleobase on the opposite strand (NTS) is also slightly unstacked.The absence of the flipped base in structures 1 and 2 suggest base flipping arises from protein induced DNA bending.
To investigate whether DNA base flipping occurs during Cas12a DNA interrogation, we employed a fluorescence-based biochemical assay.We designed DNA substrates containing single 2-aminopurine (2-AP) nucleotides at different positions relative to the PAM (Fig. 4A).This assay monitors the increase in fluorescence that occurs when 2-AP moves from a stacked, base-paired environment to an unstacked, single-stranded environment (Fig. 4A).In control reactions containing Cas12a crosslinked to DNA in the absence of guide RNA, we did not observe an increase in fluorescence relative to the 2-AP-containing double stranded DNA alone.
We next conducted fluorescence detection assays using Cas12a RNPs crosslinked to a nontargeting sequence.We observed a pronounced increase in fluorescence for substrates with 2-AP at positions 1 or 2 downstream of the PAM.Only a small increase in fluorescence was observed for substrates with 2-AP at positions 4 and 13 (Fig. 4B).These data are consistent with our structural data suggesting Cas12a flips DNA bases independent of spacer complementarity during target interrogation.A recent study of the Cas9 DNA search mechanism provided evidence for solvent exposure of bases 1 and 2 adjacent to the PAM (8).Similarity between Cas9 and Cas12a suggests that base flipping induced by DNA bending is a common interrogation strategy for Class 2 CRISPR-Cas effectors.
shifts towards the DNA by less than 1 Å globally.Helix 2 moves by ~1.3 Å relative to in structure 1.This shift is accompanied by an intermediate DNA conformation with a more pronounced bending and underwinding (45° bending and 19° underwinding versus 32° bending and 3° underwinding) than in structure 1 (Fig.2C, 3A).Structure 3 displays the largest relative rearrangement of the PI domain.Relative to structure 1 the entire PI domain of the structure 3 folds towards the DNA.The largest rearrangement is visible in helices 1-4.Helix 1, which hydrogen bonds with the PAM, moves towards DNA by ~1.5 Å. Helix 3 moves towards the DNA by ~3 Å, with concomitant motion of helix 2 by ~2.2 Å (Fig.2C).The result is a large bending and underwinding of the DNA of 68° and 41°, respectively (Fig.2C, 3A).In addition, a loop between helix 3 and 4 in the structure 3 reaches towards the PAM-distal fragment of the DNA and may form transient interactions with the DNA backbone (Fig.2C,D).These results show that movements of the PI domain correlate with DNA bending and underwinding.